Article
pubs.acs.org/molecularpharmaceutics

Functional Characterization of Apical Transporters Expressed in Rat
Proximal Tubular Cells (PTCs) in Primary Culture
Takeo Nakanishi,† Akimasa Fukushi,† Masanobu Sato,† Mayuko Yoshifuji,† Tomoka Gose,†
Yoshiyuki Shirasaka,† Kazuyo Ohe,‡,§ Masato Kobayashi,‡,§ Keiichi Kawai,‡,§ and Ikumi Tamai*,††

Department of Membrane Transport and Biopharmaceutics, Faculty of Pharmacy, ‡ Department of Quantum Medical Technology,
Faculty of Health Sciences, Institute of Medical, Pharmaceutical and Health Sciences, and §Division of Health Sciences, Graduate
School of Medical Science, Kanazawa University, Kakuma-machi, Kanazawa 920-1192 JapanS Supporting Information*

ABSTRACT: Since in vitro cell culture models often show altered apical transporter
expression, they are not necessarily suitable for the analysis of renal transport processes.
Therefore, we aimed here to investigate the usefulness of primary-cultured rat proximal
tubular cells (PTCs) for this purpose. After isolation of renal cortical cells from rat
kidneys, PTCs were enriched and the gene expression and function of apicaltransporters were analyzed by means of microarray, RT-PCR and uptake experiments.
RT-PCR confirmed that the major apical transporters were expressed in rat PTCs. Na +-dependent uptake of α-methyl-Dglucopyranoside (αMG), ergothioneine and carnitine by the PTCs suggests functional expression of Sglts, Octn1 and Octn2,
respectively. Inhibition of pH-dependent glycylsarcosine uptake by low concentration of cephalexin, which is a β-lactam
antibiotics recognized by Pepts, indicates a predominant role of high affinity type Pept2, but not low affinity type Pept1, in the
PTCs. Moreover, the permeability ratio of [14C]αMG (apical to basolateral/basolateral to apical) across PTCs was 4.3,
suggesting that Sglt-mediated reabsorptive transport is characterized. In conclusion, our results indicate that rat PTCs in primaryculture are found to be a promising in vitro model to evaluate reabsorption processes mediated at least by Sglts, Pept2, Octn1 andOctn2.
KEYWORDS: proximal tubules, kidney, renal reabsorption, SLC transporter, ABC transporter■and rat PTC NRK-52E cells.16 These artificially immortalized
cell lines are technically useful and convenient. They, however,are not necessarily appropriate to assess physiologicalreabsorption processes, because the expression levels oftransporters responsible for renal disposition of nutrients anddrugs have been altered in these cells, as compared with thoseof native kidney, freshly isolated tissues or primary-cultured
PTCs.16−18 Therefore, it remains important to establish an
appropriate cell culture model for evaluation of renal
disposition of solutes.
It has been reported that primary cultures of human PTCs
are structurally polarized, with numerous microvilli and tightjunctions at the apical side, and preserve various characteristicfeatures of proximal tubules, e.g., enzyme activities of alkaline
phosphatase and γ-glutamyl-transferase.19,20 Therefore, primary-cultured PTCs are thought to be a possible candidateof an in vitro model for studies of the mechanisms of renal
apical membrane transport, especially those involved inreabsorption. In this work, rats were chosen as a source of
PTCs because they are often used in preclinical studies for drug
development. We present functional properties of rat PTCs inprimary culture with alterations in transporter gene expression

INTRODUCTION
Renal plasma membrane transporters play a role in maintaining
nutrient homeostasis by reabsorbing various nutrients acrossthe apical membranes of proximal tubules.1 Hexoses andprotein degradation products (e.g., oligopeptides) are known tobe reabsorbed via apically expressed concentrative transporters,
Na+-dependent glucose transporters (SGLT1 and SGLT2) andH+-cotransporter PEPT2,2 in proximal tubules, particularly in
the S1 segment. We have shown that L-carnitine, an essential
substance for β-oxidation of fatty acids, is reabsorbed via
organic cation/carnitine transporter OCTN2.3,4 Absorption ofmany other nutrients is regulated by various transporters
expressed in proximal tubular cells (PTCs).5−7 On the other
hand, secretion of electrically charged compounds from tubularcells is mediated by other transporters, including organic
cation8 and anion9,10 transporters, and the ATP-driven
multidrug resistance transporters.7,11 Therefore, functional
expression of renal transporters in PTCs could be adeterminant of urinary excretion rate, residence time within
the body, and plasma concentrations of drugs in clinical use as
well as nutrients. Although kidney slices are useful for in vitro
evaluation of basolateral transport, currently available experimental models for apical transport suffer from various
disadvantages. To date, membrane transport of various
substances across the apical membranes of the proximal tubularcells (PTCs) has been examined using several cultured cell
models, e.g., human PTC HK-2,12 HKC and Caki-1 cells,13−15© 2011 American Chemical Society

Received: February 28, 2011
Revised: September 20, 2011
Accepted: October 16, 2011
Published: October 17, 2011
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Articleduring primary culture based on microarray analysis, indicatingthat the primary-cultured PTC is promising to evaluate thereabsorption processes mediated by Sglts, Pept2, Octn1 andOctn2.

■and then reverse-transcribed to cDNA with Im-Prom-IITM
reverse transcriptase (Promega, Madison, WI, USA) using anoligo(dT) primer (Invitrogen). mRNA expression of transporters was detected by RT-PCR with the respective gene
specific primers shown in Table 1. PCR was performed for 35

EXPERIMENTAL SECTION

Materials. [3H]Ergothioneine (3.7 GBq/mmol, custommade), [14C]methyl-α-D-glucopyranoside (αMG, 50 Ci/mmol), [3H]glycylsarcosine (Gly-Sar, 0.5 Ci/mmol) and[3H]mitoxantrone (2.3 Ci/mmol) were purchased from
Moravek Biochemicals (Brea, CA, USA). L-[Methyl-3H]carnitine (80 Ci/mmol) and [carboxyl-14C]inulin carboxyl(2.5 mCi/g) were purchased from American Radiolabeled
Chemicals Inc. (St. Louis, MO). [3H]Digoxin and [3H]estrone
3-sulfate were obtained from PerkinElmer Life Sciences(Waltham, MA). Percoll and collagenase (type I) werepurchased from Sigma-Aldrich (St. Louis, MO) and Wako
Pure Chemicals (Osaka, Japan), respectively. All other reagentswere of the highest grade available from Sigma-Aldrich (St.
Louis, MO), Wako Pure Chemicals (Osaka, Japan), Kanto
Chemicals (Tokyo, Japan), and Nacalai Tesque (Kyoto, Japan).
Isolation of Rat PTCs and Cell Culture. PTCs wereisolated based on the previous report 21 with several
modifications. Prior to surgery, all glassware and surgicaltools were sterilized in an autoclave, and the abdomens of rats
were shaved and cleansed with 70% (v/v) ethanol. After
surgical excision of the kidneys, renal cortical cells wereobtained by collagenase perfusion. In general, kidneys were
perfused with Ca2+-free Hanks buffer (118 mM NaCl, 4.8 mMKCl, 0.96 mM KH2PO4, 1.2 mM MgSO4, 25 mM NaHCO3, 25
mM HEPES, 2% (w/v) BSA, pH 7.4) containing 0.5 mM
EGTA at a flow rate of 8 mL/min for 10 min, followed by
perfusion with Hanks buffer containing 0.23% (wt/vol)collagenase (type I) and 2 mM CaCl2 for 15−18 min at aflow rate of 5 mL/min. All buffers were bubbled with oxygengas and maintained at 37 °C during perfusion. The collagenaseused was approximately 192 units/mg dry weight. At the end ofcollagenase perfusion, cells released from kidney were collected
into Krebs−Henseleit buffer (2.55 mM CaCl2, 25 mM HEPES,
and 2% (w/v) BSA, pH 7.4). To obtain enriched fractions ofrenal PTCs, cortical cells (1.25 mL) were layered on 8.75 mL of
an isosmotic Percoll solution (118 mM NaCl, 4.8 mM KCl,
0.96 mM KH2PO4, 1.2 mM MgSO4, 1.28 mM CaCl2, 25 mM
NaHCO3, 25 mM HEPES, 1% (w/v) BSA, 45% (v/v) Percoll,
pH 7.4) in 15 mL polycarbonate centrifuge tubes andcentrifuged for 30 min at 20000g . PTCs stayed in the upper
layer, which contained cells derived from both convoluted andstraight segments of the proximal tubules. Then, the freshly
isolated cells were suspended in Krebs−Henseleit buffer to givea concentration of 1.0 × 105 cells/mL. For primary cultures, theobtained PTCs were seeded at a density of 0.5 × 105 cells/wellon 24-well collagen-coated polystyrene tissue culture dishes(Iwaki, Tokyo, Japan). Cells were cultured in Dulbecco’s
modified Eagle’s medium/F12+GlutaMAX medium (Invitrogen Corp., Carlsbad, CA) with 10% fetal bovine serum(Hyclone; Logan, UT), 100 U/mL of penicillin and 100 mg/
mL of streptomycin, and 1 mM sodium pyruvate. The medium
was changed on days 1, 3, 5, and 7 after initial seeding. Cultures
were grown at 37 °C in a humidified incubator under an
atmosphere of 95% air, 5% CO2.
Reverse Transcription Polymerase Chain Reaction(RT-PCR) Analysis. Total RNA was prepared from primarycultured PTCs using ISOGEN (Nippon Gene, Toyama, Japan),

Table 1. Oligonucleotide Primers Used for RT-PCR
primertransporter name
Sglt1
Sglt2
Glut1
Glut2
Oat1
Oat2
Oat3
Oct1
Oct2
Oct3
Octn1
Octn2
Pept1
Pept2
Urat1

forward (5′ → 3′)

reverse (5′ → 3′)

atggacagtagcaccttgagcc
cattgtctcaggctggtactgg
tcttccaactcaaccaacca
ttggctttcactgtcttcactg
agagtcacagagccctgcat
cgctcagaattctcctccac
tcctggtgggtaccagagtc
gatctttatcccgcatgagc
catcgaggatgccgagaa
tcagagttgtacccaacgacattacctcagtgggttactttgctc
tttcgtgggtgtgctgat
gtgtggggccccaatctataccgt
cctccaaagaagtcacctccg
tgacaccgagccatgtgag

tagccccagagaagatgtctgc
ggacactgccacaatgaacacc
atacacagcagggcaggagt
gaaccagtcctgaaattagccc
gcccaggctgtagacatagc
acatccagccactccaactc
ctgcatttctgaaggcacaa
cttctgggaatcctccaagt
acagaccgtgcaagctac
tctgccacactgatgcaact
ctccgctgtgaagacgtaca
gtggaaggcgcaacaatcccatt
gtttgtctgtgagacaggttccaa
cacatctcctctcagcatggg
ggattccagccaggaagatg

cycles of denaturation at 94 °C for 30 s, annealing at 58 °C for
30 s, and extension at 72 °C for 45 s. PCR products wereanalyzed by electrophoresis in 2% agarose gel and visualizedwith ethidium bromide.
Uptake Study by Rat PTCs. For uptake study of various
radiolabeled substrates, primary-cultured PTCs were plated
onto 24-well collagen-coated tissue culture plates at a density of
0.5 × 105 cells/well. The cells were cultured for 5 days, thenwashed three times with ice-cold transport medium (130 mM
NaCl, 4.8 mM KCl, 5.6 mM D-glucose, 1.2 mM CaCl2, 1.2 mM
KH2PO4, 1.2 mM MgSO4, 25 mM HEPES, pH 7.4) before
uptake assay of radiolabeled substrates, except in the case of[3H]Gly-Sar, for which the medium pH was 6.0. In the case of
αMG uptake assay, D-glucose was removed from the transport
medium. Uptake reaction was initiated by adding transport
medium containing a radiolabeled substrate to each well, andincubation was continued at 37 °C for a certain period of time.
In inhibition studies, each inhibitor was added simultaneouslywith the radiolabeled substrate. The uptake was terminated byaddition of ice-cold transport medium, and then the cells were
washed three times with the medium to remove adhering
substrate, and lysed with 0.2 mL of 1 M NaOH. Finally, thelysate was neutralized with HCl.
Transcellular Transport Experiments for [14C]αMG.
For transcellular transport experiments, PTCs were seeded on
12-well Transwell plates (pore size, 3.0 μM) at a density of 0.5
× 105 cells/well. The cells were cultured for 5 days, thenwashed three times with transport medium (130 mM NaCl, 4.8
mM KCl, 1.2 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4,
and 25 mM HEPES, pH 7.4), and preincubated in the medium
at 37 °C for 10 min. The uptake reaction was initiated byadding transport medium containing a radiolabeled substrate tocells.
Statistical Analysis. Radioactivity was quantified with aliquid scintillation counter (Aloka, Tokyo, Japan). For protein
assay, cellular protein content was measured with a protein
assay kit (Bio-Rad) using BSA (Fraction V) as a standard. 22
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ArticleNo significant correlation in fold changes between the freshly
isolated and PTC (day 5) was observed, but fold changes
correlated well (R > 0.90) between cultured cells for 1 and 5days, suggesting that the transporter gene expression profile isunlikely affected by culture time. First of all, function of major
ABC transporters was evaluated by measuring intracellularaccumulation of digoxin (for P-glycoprotein) and mitoxantrone(for Bcrp1) in the presence of 10 μM elacridar, an inhibitor forboth P-glycoprotein and mitoxantrone, as well as estrone 3sulfate in the presence of 50 μM MK-571, an inhibitor specificto multidrug resistance associated proteins (Mrp). There wasno significant increase in intracellular accumulation of each
substrate with a respective inhibitors (Supplemental Figure 1 in
the Supporting Information). Therefore, it is not consideredthat ABC transporters affect influx of other substrates used, and
SLC transporters responsible for reabsorption of slutes inproximal tubules are further studied.
The mRNA expression of physiologically and pharmacologically important SLC transporters was examined by RT-PCR.
Figure 2 displays mRNA expression patterns of transporter

Uptake of all substrates was evaluated after correction for cellsurface-associated water content by subtraction of apparent[14C]inulin uptake. To calculate kinetic parameters for carriermediated uptake of each substrate tested, uptake rate was fitted
to eq 1 by means of a nonlinear least-squares regression analysis
using Kaleidagraph (Synergy Software, Reading, PA):(1)

where v, s, Km, and Vmax are the uptake rate of substrate (pmol
per indicated time per mg of protein), the substrateconcentration in the medium (μmol/L), the apparent
Michaelis−Menten constant (μmol/L), and the maximal
uptake rate (pmol per indicated time per mg of protein),respectively. Statistical significance of differences was determined with Student’s t test, and a P value of less than 0.05 wasconsidered statistically significant.

■

RESULTS
Cell Morphology. First of all, the morphological characteristics of PTCs in primary culture were examined under aconventional inverted light microscope. PTCs became
confluent about 5 days after isolation. The monolayer of
PTCs showed a typical epithelial morphology with cobblestone-like cells in the center of the cell islands. PTCs appeared
connected one to another (Figure 1), suggesting that they formtight junctions. This morphology is typical of proximal tubularepithelial cells.21

Figure 2. RT-PCR analysis of transporter gene expression in kidneycortex and primary-cultured PTCs. RT-PCR analysis showing PCR
products of a variety of transporters expressed in rat kidney cortex and
PTCs in primary culture (day 4). Panel (a) shows PCR products fortransporters expressed in the basolateral membrane and (b) for thoseexpressed in the apical membrane in PTCs. On day 0, PTCs wereprepared and plated on a 24-well tissue culture plate, and total cellular
RNA was extracted on day 4. PCR was performed as described in theExperimental Section after (RT+) and before (RT−) reverse
transcription. As positive and negative controls, RT-PCR was donewith total RNA from rat kidney cortex and without any RNA (RTproduct was replaced with an equal volume of H2O).

genes in PTCs cultured for 5 days and in kidney cortical tissues.
Na+-dependent glucose transporters (Sglt1 and Sglt2), organic
cation transporters (Oct1, Oct2, Oct3, Octn1 and Octn2),organic anion transporters (Oat1 and Oat3) and H +oligopeptide transporters (Pept1 and Pept2) were expressedat the mRNA level in primary-cultured PTCs. Microarray
analysis (Supplemental Table 1 in the Supporting Information)
indicates that Na+-independent facilitative glucose transporter 1(Glut1) expression was increased 4.3-fold in PTCs on day 5 ascompared to that in kidney tissue, but expression of the rest oftransporter genes shown in Figure 2, except for Oct3, wasdecreased. Among transporters with decreased expression, thelevels of Glut2 (2.35-fold), Sglt1 (4.54-fold), Octn1 (10.1-fold)and Octn2 (12.5-fold) mRNAs were relatively well maintainedin primary-cultured PTCs on day 5. mRNA expression of Oat2
and uric acid transporter (Urat1) was not detected in primarycultured PTCs.
Activity of Transporters Expressed in PTCs in Freshly
Isolated Suspension Culture. Gene expressions of SLC

Figure 1. Morphology of primary-cultured PTCs. Photograph ofprimary-cultured PTCs on day 5, taken under a standard phasecontrast light microscope. The black bar at the right bottom cornerindicates 100 μm.

mRNA Expression of Transporter Genes in PTCs in
Primary Culture. In order to examine changes of SLC
transporter gene expression during primary culture, we
employed a rat DNA microarray analysis for whole kidney
tissue, freshly isolated tubular cells (day 0) and cultured PTCs
on day 1 and day 5. Supplemental Tables 1 and 2 in the
Supporting Information show fold changes in mRNA
expression of 72 SLC and 28 ABC transporter genes that
may be involved in drug reabsorption. Numbers of SLC and
ABC transporter genes, whose fold change normalized to thatof the whole kidney tissue is less than 0.5, were 45 (62.5%) and18 (64.3%), respectively, in primary-cultured PTC on day 5.
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Articletransporters are more likely preserved in the freshly isolated
tubular cells than in the primary-cultured PTCs (Supplemental
Table 1 in the Supporting Information). At first, in order toevaluate functional expression of major apical transporters, Sglt,
Pept, Octn1 and Octn2, uptake of test compounds that
undergo reabsorption at PTCs including [ 14C]αMG (anonmetabolized substrate for Sglts), [3H]Gly-Sar (for Pepts),[3H]ergothioneine (for Octn1) and [3H]carnitine (for Octns2),
respectively, were examined in freshly isolated tubular cells insuspension culture. Uptake of [14C]αMG and [3H]carnitine
was Na+ ion dependent, and decreased to approximately 40%
and 50% in the absence of Na+, compared to the control (thepresence of Na+). However, both Na+-dependent [3H]ergothioneine uptake and H+-dependent [3H]Gly-Sar uptake
were not observed at all (data not shown). Furthermore,[14C]uric acid uptake was not inhibited by a well-establishedinhibitor of URAT1, benzbromarone, even at 100 μM (data notshown), although the expression of URAT1 was preserved
according to the microarray analysis (Supplemental Table 1 in
the Supporting Information).
Activity of Transporters Expressed in PTCs in Primary
Culture. Uptake of [14C]αMG increased linearly over 20 min
in primary-cultured PTCs in the presence of Na+, while theuptake was completely abolished in the absence of Na+ (Figure3a). Phlorizin, a potent inhibitor of Sglts, but not facilitative
glucose transporters (e.g., Gluts), significantly reduced theuptake of [14C]αMG at 10 min to about 3% of the control
uptake measured in its absence (Figure 3b). In order todetermine which Sglt, Sglt1 or Sglt2, is involved, we examinedthe effect of D-galactose, which is recognized by Sglt1 but not
by Sglt2,23,24 on the [14C]αMG uptake. Even at highconcentration (1 mM), D-galactose did not affect the[14C]αMG uptake, suggesting that Sglt2 is predominantly
responsible for the apical uptake of glucose in PTCs in primaryculture.
Similarly, [3H]Gly-Sar uptake was studied in primarycultured PTCs. Gly-Sar is an N -methylated dipeptide resistantto hydrolysis by peptidase and a well-established substrate ofboth Pept1 and Pept2. Since [3H]Gly-Sar uptake increased
linearly for up to 20 min (Figure 4a), we measured the initial
uptake rate at 10 min in the following studies. [3H]Gly-Sar
uptake by PTCs was the highest at pH 6.0 (Figure 4b). The[3H]Gly-Sar uptake at pH 6.0 was reduced to 30 and 20% in
the presence of 0.5 mM and 10 mM β-lactam antibiotics,
cephalexin, that functions as an inhibitor for both Pept1 andPept2 but shows higher affinity to Pept2 (Figure 4c).
Uptake of an Octn1 substrate, [3H]ergothioneine, wasdependent on temperature (Figure 5a). The uptake of[3H]ergothioneine was decreased to 25% and 30% in theabsence of Na+ and in acidic transport medium (pH 6.0),
respectively (Figure 5b). Uptake of [3H]ergothioneine by
primary-cultured PTCs was saturated at concentrations over
200 μM (Figure 5c) and consisted of a single component withKm of 7.4 ± 9.3 μM and Vmax of 71.7 ± 21.1 pmol/mg ofprotein/5 min (Figure 5c). Furthermore, [3H]carnitine uptake
was studied to evaluate functional expression of Octn2.
[3H]Carnitine uptake increased linearly over 10 min inprimary-cultured PTCs (Figure 6a). [3H]Carnitine uptake
decreased to 65% in the absence of Na +. In addition,[3H]carnitine uptake was reduced by 65% and 48% in thepresence of excess amounts of unlabeled carnitine (500 μM)
and arginine (500 μM), respectively, suggesting that Octn2 isinvolved in carnitine uptake by PTCs (Figure 6b).
Transcellular Transport of [14C]αMG in PrimaryCultured PTCs. To determine whether primary-cultured
PTCs are suitable for evaluation of transcellular transport, we
examined the trans-epithelial electrical resistance (TEER) of
PTCs cultured on a Transwell insert. At day 6 after seeding,
TEER reached a plateau and remained constant at about 140
Ω·cm2 in culture for up to a further 6 days (data not shown).
Then, the transcellular permeability of [14C]αMG in primarycultures of PTCs on Transwells was evaluated. Apical (AP) tobasolateral (BL) (Figure 7a) and BL to AP (Figure 7b)transcellular transport of [14C]αMG across a monolayer of
PTCs was measured for 30 min. The AP to BL and the BL to
AP permeability values of [14C]αMG were 10.7 ± 0.39, and2.46 ± 0.03 ( × 10−5 cm/s), respectively (Table 2). These rates
were significantly higher than paracellular leakage of [3H]mannitol (1.38 ± 0.29 × 10−5 cm/s). The AP-to-BL
permeability of [14C]αMG was significantly reduced in theabsence of Na+ (Table 2). The AP-to-BL permeability ratio of[14C]αMG in transporter buffer containing Na+ over thatwithout Na+ was 1.58, whereas the corresponding ratio for BLto-AP permeability was 0.53. Furthermore, the permeability
ratio of [14C]αMG determined as the value of (AP-to-BL)/(BL-to-AP) in the presence of Na+ was 2.97-fold greater thanthat in the absence of Na+. These results demonstrate that the

Figure 3. [14C]αMG uptake by primary-cultured PTCs. (a) Timecourse of [14C]αMG (20 μM) uptake by primary-cultured PTCs.
Uptake was measured at pH 7.4 with (closed symbols for control) or
without (open symbols) Na+ for 20 min. (b) Effect of Sglts inhibitorson [14C]αMG uptake by primary-cultured PTCs. Uptake of[14C]αMG (20 μM) was measured at pH 7.4 and 37 °C for 10 min
with (D-galactose (grey column), or phlorizin (white column), or
without (closed column; control) inhibitors. Each point or bar
represents the mean value with SEM (n = 3) of uptake after
subtraction of [14C]inulin uptake. * indicates a statistically significant
difference from the control by Student’s t test (p < 0.05).

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ArticleFigure 5. [3H]Ergothioneine uptake by primary-cultured PTCs. (a)Time course of [3H]ergothioneine (5 μM) uptake by primary-cultured
PTCs. Uptake was measured at pH 7.4 and 37 °C (closed column;
control) or 4 °C (open column) for 20 min. (b) Effect of Na+ and pH
on [3H]ergothioneine uptake by primary-cultured PTCs. Uptake of[3H]ergothioneine by primary-cultured PTCs was measured at pH 7.4
and 37 °C for 5 min. (c) Concentration dependence of [3H]ergothioneine uptake by primary-cultured PTCs. The data were fitted
to the Michaelis−Menten equation by a nonlinear least-squares
regression analysis, as described in the Experimental Section. Inset:
The uptake was analyzed by means of the Eadie−Hofstee plot. Eachpoint or bar represents the mean ± SEM (n = 3), after subtraction of
the uptake of [14C]inulin.

Figure 4. [3H]Gly-Sar uptake by primary-cultured PTCs. (a) Timecourse of [3H]Gly-Sar (20 μM) uptake by primary-cultured PTCs.
Uptake was measured at pH 7.4 and 37 °C (closed column; control)
or 4 °C (open column) for 20 min. (b) pH dependence of [3H]GlySar uptake by primary-cultured PTCs was measured. Uptake of[3H]Gly-Sar (1 μM) was measured at various pH values (range 5.5−8.0) for 10 min at 37 °C. (c) Inhibitory effect of amino acid, dipeptide
and β-lactam antibiotics on [3H]Gly-Sar uptake by primary-cultured
PTCs. Uptake of [3H]Gly-Sar (1 μM) by primary-cultured PTCs wasmeasured at pH 7.4 and 37 °C for 10 min with (open column) or
without (closed column; control) inhibitors. Each point or bar
represents the mean value with SEM (n = 3) after subtraction of[14C]inulin uptake. * indicates a statistically significant difference fromthe control by Student’s t test (p < 0.05).

not been established. In the present study, PTCs cultured onplastic or semipermeable membranes were assessed theaptitude for this purpose, and we were able to show thatseveral important apical uptake transporters are functionally
maintained in these cells. Species differences, to date, have beenreported in transporter expression between humans and rats.
For example, hOCT1 is little expressed, whereas significant
expression of rOct1 is detected in the basolateral membranes. 25
In the case of OAT2, it is expressed at the basolateral
membranes and plays a role in elimination of anioniccompounds from blood to PTCs in humans,26 while rOat2
was detected in the apical surface of the tubules in the
medullary thick ascending limb of Henle’s loop,27 and may becontributable to reabsorption of the compounds. Despite suchspecies differences, this PTC model cell can be usable toexamine activities of the common transporters involved inreabsorption in both species. Hence, Sglts, Pepts, Octn1/2 andpresent cultured cell model adequately reflects Sglt function in
AP to BL transport.

■

DISCUSSION
There is an increasing demand for methods to evaluate theactivities of transporters responsible for renal reabsorption ofdrugs and nutrients in order to predict in vivo renal disposition.
Apparent activity of transporters in the basolateral membrane
can be measured by using kidney slices, but an effective method
for measuring the activities of apical membrane transporters has
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ArticleFigure 7. Effect of Na+ on [14C]αMG trans-epithelial transport acrossprimary-cultured PTC monolayer. (a) Transcellular transport (in the
AP to BA direction) and (b) transcellular transport (in the BA to AP
direction) of [14C]αMG and [3H]mannitol. [14C]αMG was added tothe apical side of a primary-cultured PTC monolayer with (closed
symbol, control) or without (open symbol) Na+. Closed diamonds
show transcellular transport of [3H]mannitol as a paracellular marker.
For 30 min at 37 °C, [14C]αMG and [3H]mannitol translocated into
the basolateral side (a) and the apical side (b) were measured. Theirapparent permeability coefficients were calculated as described in theExperimental Section (Table 2). Each point represents the mean ±SEM (n = 3). * and † indicate a statistically significant difference intranscellular transport in Na+-free buffer vs that in the control (with
Na+), and in transcellular transport in the direction of AP to BA vs BA
to AP, measured in the buffer with Na+, by Student’s t test (p < 0.05),
respectively.

Figure 6. [3H]Carnitine uptake by primary-cultured PTCs. (a) Timecourse of [3H]carnitine uptake by primary-cultured PTCs. Uptake wasmeasured at pH 7.4 and 37 °C (closed column; control) or 4 °C
(open column) for 20 min. (b) Effect of Na+ and inhibitors on[3H]carnitine uptake by primary-cultured PTCs. Uptake of [ 3H]carnitine (12 nM) by primary-cultured PTCs was measured at 37 °C
for 10 min with (closed column) or without (open column) Na +. Eachpoint or bar represents the mean ± SEM (n = 3) after subtraction of
the uptake of [14C]inulin. * indicates a statistically significant
difference from the control (with Na+ and without inhibitors) byStudent’s t test (p < 0.05).

Urat1 were chosen for the assay in the present study because
species difference has not been seen so far.
We initially examined changes of mRNA expression by
employing a rat DNA microarray assay. In many genes, mRNA
expression seemed to be preserved in freshly isolated PTCs
(day 0). However, function of transporters for ergothioneine,uric acid and oligopeptides was unlikely consistent to theirreabsorption literally described. Since freshly isolated cells did
not attach well to plastic plates, uptake had to be done insuspension culture of the PTCs. Therefore, we were not able todistinguish apical from basolateral uptake of the test
compounds. That led us to culture PTCs on the plates fordays. mRNA expression of major transporters expressed in
PTCs, but Oat2 and Urat1, were detected under the culture
conditions (Figure 2). To date, the absence of gene expressionof transporters has been reported in several proximal tubulederived mammalian cell culture models. For example, Caki-1
cells lack mRNA expression of organic cation transporters,
including OCT1, OCT2 and OCTN1, and organic aniontransporters, including OAT1, OAT2 and OAT3.15,28 Moreover, Oat1, Oat2, Oat3, Oatp1a1 and Oatp1a3 were notdetected in NRK-52E cells, a rat proximal tubular cell line, in
RT-PCR assay.16 Swine PTCs, LLC-PK1, which are often used
to evaluate drug transport through proximal tubular cells, alsolack several organic anion transporters18 and proximal brushborder enzymes, compared with those in isolated and primarycultured PTCs.17 Taking these findings together, it isTable 2. Effect of Na+ on [14C]αMG Permeability acrossPrimary-Cultured PTC Monolayera
A to B
B to A
A to B/B to A

control

Na+-freecontrol/Na+-free

10.68 ± 0.39
2.46 ± 0.03
4.34

6.76 ± 1.05
4.64 ± 0.23
1.46

1.58
0.53

a

Each value is the mean ± SEM (×10−5 cm/s). [14C]αMG (2.5 μM)
was added to the apical side of primary-cultured PTC monolayer withor without Na+. After 30 min at 37 °C, [14C]αMG (2.5 μM)
transported into basal side was measured. † indicates a statisticallysignificant difference (AP-to-BL vs. BL-to-AP). * indicates astatistically significant difference (control vs Na+-free).

considered that expression of major transporters was relatively
well preserved in the primary-cultured PTCs. Since previous
reports showed that medium composition might influence theexpression and activity of brush-border enzymes,29,30 manipulating medium composition may help to understand alterationof gene expression in primary-cultured PTCs. Future study iswarranted to compare protein expression levels with functional
activity in the primary-cultured PTCs to accurately describe theusefulness of this cell model.
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ArticleUptake studies with a variety of endogenous substrates wereconducted (Figures 3 through 6). It has been generally
accepted that uptake reflects reabsorption processes since paminohippuric acid by PTCs plated on culture dishes wasshown to correspond to its reabsorption from the apical lumenof proximal tubules.31 In the proximal tubules, most of glucose
filtrated by glomerulus is reabsorbed via Sglts, which areessential to maintain the plasma glucose level. Both Sglt1 (highaffinity/low capacity) and Sglt2 (low affinity/high capacity) are
expressed in the brush-border membranes in PTCs. 23,24
Although phlorizin is a common inhibitor of both Sglts, theydiffer in binding affinity for D-galactose; i.e., D-galactose
preferentially inhibits Sglt1 over Sglt2.23,24 As shown in Figure
3b, the finding that αMG uptake was inhibited by phlorizin but
not by D-galactose implies that Sglt2 more dominantly
contributes to reabsorption of glucose. These results agreewith the previous reports of phlorizin-sensitive Na+-dependent
glucose transport at the apical side in PTCs.32
Pept1 and Pept2 are pH-dependent peptide transporters, and
Pept2 is predominantly expressed in the brush-border
membranes of PTCs.2 Figure 4 suggests that Pepts arefunctional, because [3H]Gly-Sar uptake by PTCs was induced
by an inward H+ gradient. Cephalexin has greater affinity to
Pept2 with a Ki (inhibition constant) of 0.049 mM (the Ki ofPept1 is approximately 4.5 mM).33 Therefore, the 70%
reduction of [3H]Gly-Sar uptake by cephalexin at theconcentration of 0.5 mM is accounted for by its competitive
inhibition of high-affinity-type Pept2, but not low-affinity-typePept1, implying that this model reflects well the in vivo
oligopeptide reabsorption via Pept2 at the proximal tubules.
We have originally identified Octn1 as a pH-dependent
organic cation transporter expressed in PTCs.18 Octn1 has
been reported to transport ergothioneine in a Na+ and pHdependent manner.34,35 Since Octn1 is localized on the apicalmembrane of PTCs,36 we examined whether Octn1 function
was retained in primary-cultured PTCs. Na +- and pHdependent ergothioneine uptake was observed (Figure 5b).
Kinetic analysis of the initial uptake of ergothioneine by rat
Octn1 showed a linear Eadie−Hofstee plot with a Km value of
7.4 μM (Figure 5c), which is close to our previously reported
value (4.64 μM),35 suggesting that Octn1 is functionallyexpressed at the surface of the primary-cultured PTCs. In
addition, Octn2-mediated carnitine transport in the present
system was examined, since Octn2 is responsible forreabsorption of carnitine.3,4,37 Although carnitine is transported
by amino acid transporter ATB0,+ in an Na+-dependent
manner,38 our data show that carnitine uptake by PTCs was
not inhibited by arginine, a typical substrate of ATB0,+ (Figure6b). Therefore, it is considered that carnitine uptake in PTCs inprimary culture reflects Octn2 function. However, carnitine
uptake was not completely abolished by removal of Na + (Figure6b), hence other transporter system(s) may play a role incarnitine reabsorption.
Finally, we examined whether primary-cultured PTCs can beused to evaluate transcellular transport of small molecules,
corresponding to reabsorption. In the presence of Na +, AP-toBL permeation of [14C]αMG was higher than BL-to-AP
permeation (Table 2). Furthermore, AP-to-BL transport of[14C]αMG in the presence of Na+ was higher than that in theabsence of Na+, while a substantial amount of AP to BL
transport of [ 14C]αMG was preserved in the Na +-free
condition, compared with paracellular leakage evaluated interms of the permeability of mannitol. This significant Na+-

independent transport was inconsistent with our results in the
uptake experiments (Figure 3a), where [14C]αMG was almost
completely taken up in a Na+-dependent manner. A possible
explanation is that Na+-independent glucose transporter Glut
may play a role in AP-to-BL transcellular transport of[14C]αMG, although such a discrepancy between uptake andtransport experiments in the PTCs used in the present study
should be clarified in detail if this system is to be further
developed. The observation that BL-to-AP permeability of[14C]αMG in the absence of Na+ was greater than that in thepresence of Na+ could be explained by reuptake of permeated[14C]αMG into the AP side by Sglt expressed in the apicalmembranes, resulting in a decreased appearance of αMG on theapical side, which could not occur in Na+-free buffer. In thepresent study, we did not examine transcellular transport ofother substrates, and further study needs to be done tocharacterize transcellular transport for other molecules in PTCs
in order to assess the usefulness as a model.
In conclusion, PTCs in primary culture maintained functional expression of major transporters, such as Sglt2, Pept2,
Octn1 and Octn 2, involved in renal reabsorption of naturalnutrients and drugs, and could be a promising model forevaluation of apical uptake. Further cytohistochemical study forthe transporter protein should validate reliability of this in vitro
cell culture model to evaluate not only renal disposition ofdrugs, but also drug−drug and drug−food interactions in therenal reabsorption process.

■

ASSOCIATED CONTENT
S Supporting Information*
Supplemental Table 1 presenting changes of SLC transporter
gene expression in PTCs, Supplemental Table 2 presentingchanges of ABC transporter gene expression in PTCs, andSupplemental Figure 1 depicting intracellular accumulation of
ABC transporter substrates. This material is available free ofcharge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author*Department of Membrane Transport and Biopharmaceutics,
Faculty of Pharmacy, Institute of Medical, Pharmaceutical and
Health Sciences, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan. E-mail: tamai@p.kanazawa-u.ac.jp.

■

ABBREVIATIONS USED
PTC, proximal tubular cells; SLC, solute carrier; ABC, ATPbinding cassette; αMG, α-methyl-D-glucopyranoside; AP,apical; BP, basolateral; SGLT/Sglt, Na+-dependent glucose
transporters; Gly-Sar, glycylsarcosine; RT-PCR, reverse transcription polymerase chain reaction; OCT/Oct, organic cationtransporter; OAT, organic anion transporter; PEPT/Pept, H +oligopeptide transporter; TEER, trans-epithelial electricalresistance■

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